Secretion In Cortical Collecting Duct Research Articles

Cell-specific regulation of L-WNK1 by dietary K.

Flow-induced K(+) secretion in the aldosterone-sensitive distal nephron is mediated by high-conductance Ca(2+)-activated K(+) (BK) channels. Familial hyperkalemic hypertension (pseudohypoaldosteronism type II) is an inherited form of hypertension with decreased K(+) secretion and increased Na(+) reabsorption. This disorder is linked to mutations in genes encoding with-no-lysine kinase 1 (WNK1), WNK4, and Kelch-like 3/Cullin 3, two components of an E3 ubiquitin ligase complex that degrades WNKs. We examined whether the full-length (or "long") form of WNK1 (L-WNK1) affected the expression of BK α-subunits in HEK cells. Overexpression of L-WNK1 promoted a significant increase in BK α-subunit whole cell abundance and functional channel expression. BK α-subunit abundance also increased with coexpression of a kinase dead L-WNK1 mutant (K233M) and with kidney-specific WNK1 (KS-WNK1), suggesting that the catalytic activity of L-WNK1 was not required to increase BK expression. We examined whether dietary K(+) intake affected L-WNK1 expression in the aldosterone-sensitive distal nephron. We found a paucity of L-WNK1 labeling in cortical collecting ducts (CCDs) from rabbits on a low-K(+) diet but observed robust staining for L-WNK1 primarily in intercalated cells when rabbits were fed a high-K(+) diet. Our results and previous findings suggest that L-WNK1 exerts different effects on renal K(+) secretory channels, inhibiting renal outer medullary K(+) channels and activating BK channels. A high-K(+) diet induced an increase in L-WNK1 expression selectively in intercalated cells and may contribute to enhanced BK channel expression and K(+) secretion in CCDs.

Protein Kinase C Inhibits ROMK1 Channel Activity via a Phosphatidylinositol 4,5-Bisphosphate-dependent Mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.

Regulation of the apical Cl-/HCO-3 exchanger pendrin in rat cortical collecting duct in metabolic acidosis.

Pendrin is an apical Cl(-)/OH(-)/HCO(3)(-) exchanger in beta-intercalated cells (beta-ICs) of rat and mouse cortical collecting duct (CCD). However, little is known about its regulation in acid-base disorders. Here, we examined the regulation of pendrin in metabolic acidosis, a condition known to decrease HCO(3)(-) secretion in CCD. Rats were subjected to NH(4)Cl loading for 4 days, which resulted in metabolic acidosis. Apical Cl(-)/HCO(3)(-) exchanger activity in beta-ICs was determined as amplitude and rate of intracellular pH change when Cl was removed in isolated, microperfused CCDs. Intracellular pH was measured by single-cell digital ratiometric imaging using fluorescent pH-sensitive dye 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-AM. Pendrin mRNA expression in kidney cortex was examined by Northern blot hybridizations. Expression of pendrin protein was assessed by indirect immunofluorescence. Microperfused CCDs isolated from acidotic rats demonstrated approximately 60% reduction in apical Cl(-)/HCO(3)(-) exchanger activity in beta-ICs (P < 0.001 vs. control). Northern blot hybridizations indicated that the mRNA expression of pendrin in kidney cortex decreased by 68% in acidotic animals (P < 0.02 vs. control). Immunofluorescence labeling demonstrated significant reduction in pendrin expression in CCDs of acidotic rats. We conclude that metabolic acidosis decreases the activity of the apical Cl(-)/HCO(3)(-) exchanger in beta-ICs of the rat CCD by reducing the expression of pendrin. Adaptive downregulation of pendrin in metabolic acidosis indicates the important role of this exchanger in acid-base regulation in the CCD.

Maturation of HCO3- transport in rabbit collecting duct

Newborn rabbits maintain a state of hypochloremic metabolic alkalosis with plasma HCO3- concentrations generally exceeding 27 mM. The large amounts of potential net base in mother's milk probably contribute to the generation of this alkalosis. We surmised that immature handling of H+ or HCO3- by the neonatal collecting duct helps maintain this alkalosis. Net HCO3- transport was measured in perfused collecting ducts taken from maturing rabbits, in solutions simulating adult rabbit plasma ultrafiltrate and then in presence of Cl(-)-free bathing solution. Cortical collecting ducts (CCD) from newborn and 4-wk-old rabbits failed to secrete HCO3- under baseline conditions and could not be stimulated to secrete HCO3- in Cl(-)-free bath. Neonatal segments perfused at very slow flow rates showed significant HCO3- absorption; inhibition of HCO3- secretion by removal of luminal Cl- revealed a substantial HCO3- absorptive flux. Segments from 6-wk-old and mature animals secreted net HCO3- and generally showed more than a fivefold increase in HCO3- secretion after Cl- removal from the bath. Outer medullary collecting ducts (OMCD) from newborn rabbits absorbed HCO3- at rates approaching that of mature segments. We conclude that relatively high rates of HCO3- absorption in OMCD and lack of HCO3- secretion in CCD may contribute to the metabolic alkalosis of the neonatal rabbit.